JP3220420B2 - Pentapeptide, its production method and its use - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel peptide having utility as a food or pharmaceutical and a method for producing the same, and further relates to a hypotensive agent containing the peptide as an active ingredient.

[0002]

2. Description of the Related Art Hypertension is broadly classified into those in which the cause of the increase in blood pressure is obvious (symptomatic hypertension) and those in which the cause is unknown (essential hypertension). Although symptomatic hypertension can cure hypertension by curing the underlying disease, direct treatment for the cause is difficult with essential hypertension. On the other hand, renin-angiotensin system (hereinafter, referred to as
Abbreviated as RA system. ) Is considered to be one of the important factors of essential hypertension.
Attempts have been made to regulate essential hypertension by regulating the RA system by inhibiting the activity of angiotensin converting enzyme (hereinafter abbreviated as ACE), which plays a central role in the system.

[0003] As a substance having such ACE activity inhibition, an L-proline derivative or a compound based thereon is known in the case of a synthetic compound, and a substance derived from a snake venom in the case of a substance derived from a natural product. Bradykinin enhancers, six types of hexapeptides derived from collagenase digest of gelatin, and peptides of tryptic digest of bovine casein are known. In particular, in recent years, natural products derived from natural products that are superior in terms of safety compared to synthetic compounds that remain a concern in terms of side effects, among those found in foods that we take daily, have low toxicity. Is expected to be a highly safe antihypertensive agent.

[0004]

However, ACE inhibitors found in natural products are present in small amounts.
For peptides, the preparation method is extremely difficult.
The present invention has been made in view of such a situation, and provides an antihypertensive agent with low toxicity and high safety by searching for an ACE inhibitor with few side effects using natural products, especially foods considered to be high in safety. It is intended to do so.

[0005]

Means for Solving the Problems The present inventors have found that there is a substance having ACE inhibitory activity in a composition obtained by hydrolyzing laver protein with pepsin. 8-332270), this time,
-Alanine (Ala)-lysine (Lys)-tyrosine (Tyr)-serine (Ser)-tyrosine (Tyr)-
The present inventors have identified the peptide as having an OH amino acid sequence and completed the present invention.

That is, the present invention provides the following formula: H-Ala-L
The present invention relates to a novel pentapeptide having an L-form amino acid sequence represented by ys-Tyr-Ser-Tyr-OH. Also, the present invention decomposes seaweed into pepsin,
The present invention relates to a method for producing the pentapeptide, which is characterized in that the pentapeptide is separated and purified. Furthermore, the present invention relates to a hypotensive agent comprising the novel pentapeptide as an active ingredient.

The H-Ala-Lys-Tyr-S of the present invention
The peptide consisting of er-Tyr-OH is a novel peptide not described in the literature, and is produced by hydrolyzing laver protein with pepsin. It can also be produced by synthesizing by applying conventional means of peptide synthesis.

In the case of producing seaweed protein by hydrolyzing it with pepsin, a predetermined amount of pepsin is added to seaweed and the reaction is carried out at about 30 to 60 ° C. for 1 to 48 hours. In the hydrolyzed solution, other peptides and components other than the peptides other than the peptide of the present invention are also present, and are isolated therefrom. Isolation is performed by separating the hydrolyzed solution by a known operation such as centrifugation, and then applying extraction, concentration, drying, etc., and then differences in adsorption affinity for various adsorbents and differences in solubility in various solvents. It is preferable to isolate the target substance by applying means utilizing the difference in elution rate based on the size of the molecule, the dialyzability from the solution or the difference in dialysis rate. These methods may be used alone as needed, or combined in any order, and applied repeatedly.

When the peptide of the present invention is produced by a synthetic method, one of two types of fragments which are bisected at an arbitrary position of the peptide bond by a method usually used for peptide synthesis, that is, a liquid phase method or a solid phase method, is used. When a material having a corresponding reactive carboxyl group and a material having a reactive amino group corresponding to the other fragment are condensed using a carbodiimide method, an active ester method, or the like, and the resulting condensate has a protective group. , By removing the protecting group. The pentapeptide of the present invention has an ACE inhibitory activity and is useful as a hypotensive agent with few side effects.

[0010]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto. (Production Example 1) Peptide H-Ala-Lys by pepsin decomposition of laver
-Preparation of Tyr-Ser-Tyr-OH

[0011] 50 g of dried seaweed finely pulverized to 35 mesh by a high-speed pulverizer is mixed with an HCl-KCl buffer (PH =
2.0) It becomes turbid in 950 ml, and pepsin (manufactured by Amano Pharmaceutical)
2 g was added and reacted at 50 ° C. for 24 hours with stirring. The obtained decomposition solution was neutralized with a 1N solution of NaOH, and 1400
The mixture was centrifuged at 0 rpm for 20 minutes, and the supernatant was filtered with a glass filter. Thereafter, the enzyme was inactivated by immediately holding in a boiling bath for 20 minutes to obtain a decomposed product of laver protein.

Next, the obtained seaweed protein hydrolyzate is
Bio-Rad Dowex-50 (H ⁺ )
Load onto a column (4.5 cm x 43 cm), wash with 5 liters of distilled water, elute the adsorbed peptide with 2N ammonia water, remove ammonia by evaporation, freeze-dry, and extract from seaweed A peptide mixture was obtained.

Next, the obtained peptide mixture was added to 0.05
% Trifluoroacetic acid aqueous solution
APCELPAK UG-120 (φ10mm × 250
mm) (manufactured by Shiseido Co., Ltd.), and analyzed by linear gradient HPLC using 25% acetonitrile containing 0.05% trifluoroacetic acid at a flow rate of 4 ml / min, for 52.8 to 53.4 min. The peak was collected, lyophilized, and analyzed for amino acid sequence and amino acid composition by hydrolysis with hydrochloric acid using a protein sequencer. As a result, H-Ala-Lys-Tyr-Ser-Tyr-O
A peptide having the structure of H was obtained.

(Production Example 2) Peptide H-Ala-Lys-Tyr-S by solid phase method
Preparation of er-Tyr-OH

The peptide was synthesized by a solid phase method using an automatic peptide synthesizer manufactured by Applied Biosystems. As the solid phase alone, a resin obtained by chloromethylating a styrene-divinylbenzene copolymer (polystyrene resin) was used. First, according to the amino acid sequence of the peptide, a C-terminal tyrosine was reacted with a chloromethyl resin according to a conventional method to obtain a peptide-bound resin. The amino acid at this time is t-butoxycarbonyl (hereinafter “t-butoxycarbonyl”).
-Boc) group protected t-Boc amino acid was used.

Next, this peptide-bonded resin is suspended in a mixture of ethanediol and thioanisole, and
After stirring for 0 minutes, trifluoroacetic acid was added under ice cooling, and the mixture was further stirred for 10 minutes. Trifluoromethanesulfonic acid was added dropwise to the mixture, and the mixture was stirred at room temperature for 30 minutes, and anhydrous ether was added to precipitate the product. The precipitate was separated and washed with anhydrous ether several times. And dried. The unpurified synthetic peptide thus obtained was dissolved in distilled water, and then, using a reversed-phase column C18,
Purification was attempted by linear gradient HPLC using 50% acetonitrile containing 0.1% trifluoroacetic acid. The eluted fraction detected at an ultraviolet wavelength of 220 nm and exhibiting the maximum absorption was collected and freeze-dried to obtain a target peptide synthesized product. TO for the obtained peptide
FIG. 1 shows the results of F-MS.

Test Example 1 Measurement of ACE Inhibitory Activity H-Ala-L of Preparation Example 2 dissolved in a test tube at a predetermined concentration.
50 μl of a peptide showing the structure of ys-Tyr-Ser-Tyr-OH was added, and then 12.5 L-hyprylhistidylleucine (manufactured by Peptide Research Institute) was used as an enzyme substrate.
Dissolve in a borate buffer (pH = 8.3) containing 1.0 M sodium chloride to a concentration of mM, add 100 μl to the above test tube, and finally add distilled water to 25 mU / ml. Add 100 μl of the dissolved ACE solution and add 3
The reaction was performed at 7 ° C. for 1 hour. Then, 0.5N of HCl
The reaction was stopped by adding 250 μl of the solution and left for 5 minutes.
1.5 ml of ethyl acetate was added to the tube wall while being transmitted, and after vigorous stirring, centrifugation (3000 rpm, 10 minutes) was performed, and 0.5 ml of the upper layer (ethyl acetate layer) was collected. After evaporating the ethyl acetate for 3 minutes, the generated hippuric acid was dissolved in 3 ml of 1.0 M sodium chloride, and the absorbance was measured at 228 nm.

Inhibition rate (%) = Es is the absorbance of the sample, Ec is the value obtained by adding distilled water instead of the sample, and Eb is the value obtained by adding a reaction stop solution in advance to react.
{(Ec−Es) / (Ec−Eb)} × 100. The activity IC ₅₀ value for ACE inhibition is the sample concentration required to inhibit ACE by 50%. As a result, the IC ₅₀ of the peptide was 1.2 × 10 ⁻⁶ M.

(Test Example 2) Hypotensive effect upon administration to rats A 15-week-old male spontaneously hypertensive rat (SHR) was purchased from Japan SLC, Inc., and after pre-breeding for 2 weeks, the systolic blood pressure was 190 mmHg. Using the above six animals as one group, the H-Ala-Lys-Tyr-Ser-
A peptide having a Tyr-OH structure was suspended in a 0.5% aqueous solution of CMC and orally administered at 300 (mg / kg po) using a metal probe.

The blood pressure was measured by a tail-cuf using a non-invasive tail arterial blood pressure measurement device (Muromachi Kikai, Model MK-1030).
According to Method f, before administration, 1 hour after administration, 2 hours, 4 hours,
Six-hour, eight-hour SHR tail systolic blood pressure (SB
P), mean blood pressure (MBP), diastolic blood pressure (DBP), and pulse measurement were performed five times at each measurement time, and the highest and lowest values of the obtained measurement values were discarded, and the average value of the three measurements was used for each measurement. Time measurements were taken. As a result, it was confirmed that only the systolic blood pressure significantly decreased 4 hours after the administration. In addition, the pulse rate also decreased after 2 hours, and tended to decrease thereafter. Table 1 shows these results.

[0021]

[Table 1]

In general, when a hypotensive drug is administered, side effects in which tachycardia is induced with a decrease in blood pressure may be observed, but the present peptide was found to have few such side effects.

(Preparation example) Preparation of anti-hypertensive infusion solution 50 mg of the present peptide prepared in Production Example 2, 9 g of sodium chloride, 5 g of chlorobutanol, and 1 g of sodium hydrogen carbonate
g was dissolved in 1000 ml of distilled water, and this was dispensed into two drip bottles to obtain an anti-hypertensive infusion.

[0024]

As described above, the peptide of the present invention has an ACE inhibitory activity and exhibits a blood pressure lowering action derived therefrom, and is therefore useful as a blood pressure lowering agent. This peptide can also be produced by enzymatic decomposition of laver or a synthetic method.

[Brief description of the drawings]

FIG. 1 is a chart of TOF-MS analysis of a peptide of the present invention.

Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 7/06 C12P 21/06 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. The following formula: H-Ala-Lys-Tyr-S
A novel pentapeptide having a peptide structure based on the L-amino acid sequence represented by er-Tyr-OH. 2. The method for producing a pentapeptide according to claim 1, wherein the laver is pepsin-decomposed and separated and purified. 3. The following formula: H-Ala-Lys-Tyr-S
An antihypertensive agent comprising, as an active ingredient, a novel pentapeptide having a peptide structure based on the amino acid sequence of L-form represented by er-Tyr-OH.